There is a growing recognition that mild traumatic brain injury (mTBI) or concussion is a major health issue, with over 2 million cases each year in the United States. Moreover, there is increasing concern that there may be a period of vulnerability after one mTBI, during which time a second mTBI may induce greatly exaggerated pathophysiological effects. We have used a multidisciplinary approach to generate experimental in vitro and in vivo models that replicate the mechanical loading conditions of mTBI in humans. With these models, we have recently identified that diffuse axonal injury (DAI) is a key pathology of single mTBI. In addition, we have shown that microtubule damage as well as dysregulation of sodium channels on axons following traumatic injury are watershed events contributing to axonal vulnerability, dysfunction and degeneration. Moreover, we have generated promising preliminary data indicating the utility of novel neuroimaging and serum biomarkers for the detection of brain damage in mTBI. Here, we propose to use these models to 1) investigate novel diagnostic techniques to evaluate axonal pathology in single and repetitive mTBI, and 2) explore the mechanistic roles of axonal cytoskeletal changes that may induce enhanced vulnerability that accounts for exacerbated damage with a repeat mTBI. Innovation: Success of the proposed studies could lead to 1) new diagnostic criteria to identify mTBI pathologies, 2) identify new pathologies and mechanisms of single and repetitive mTBI, and 3) reveal new therapeutic targets.